1. Field of the Invention
The invention relates to screening assays. More specifically, the invention relates to screening assays for enzyme inhibitors using enzyme complementation.
2. Background and Related Disclosures
The impending resolution of the human and other genomes has provided an extensive repertoire of protein targets for modulation of mammalian physiology. The ability to identify the genes of the mammalian genomes has greatly expanded the need to determine the function of the proteins and the manner in which proteins interact to fulfill their function. At the same time, combinatorial approaches have greatly expanded the number of candidate compounds that may serve as drugs. Due to the large number of permutations and combinations, there is a particular need for rapid, accurate assays that do not require numerous steps, complicated protocols, and expensive reagents and equipment for their performance.
There is a rich literature of protocols and reagents for performing analyte determinations. For the most part, with haptens that are monoepitopic and frequently with antigens that are polyepitopic, competitive assays are employed. That is, the analyte and a modified analyte compete for a binding protein. Binding of the modified analyte with the binding protein results in signal modulation, where the modulation is related to the amount of analyte present in the sample.
For the most part, the binding protein has been polyclonal or monoclonal antibodies. These binding proteins are preferred because of their high affinity and specificity. In addition, the off-rate of the analyte or modified analyte is relatively slow, with the equilibrium under the conditions of the assay being driven toward the binding complex, rather than toward the unbound analyte. The success of the assay has been dependent on obtaining an extended dynamic range and clean results due to the high binding affinity of the antibodies.
Where one is interested in determining the binding of an agent to a target, various protocols may be employed. Where the target has a lower affinity than an antibody, protocols requiring an antibody in competition with the target will generally not be useful. Since the binding of the agent is reversible, the agent would distribute itself between the antibody and the target. Unless the target and the antibody have comparable binding affinities, the agent would primarily bind to the one that has the higher binding affinity, providing a very small dynamic range.
Alternatively, one may provide a modified agent, where the agent is coupled to a detectable label, and allow for a competition between the modified agent and the agent for the target. One may then isolate the complex of the target with the modified agent and detect the level of binding to the target, which can be correlated to the binding affinity of the agent to the target. This requires that a different modified agent must be prepared for each agent.
In particular for enzyme inhibitor assays, one approach has been to develop antibodies to the product and monitor the formation of product in the presence of an inhibitor. This approach is exemplified by kinases and phosphatases, where antibodies are made that are specific for a phosphorylated or dephosphorylated product. This approach requires that a different substrate be prepared for each enzyme and different antibodies be prepared for each product. In fact, the preparation of antibodies to the phosphorylated or dephosphorylated product that distinguish between the product and the much greater amount of substrate has proven problematical.
Previous studies have shown that [3H]-staurosporine (Herbert et al, BBRC, 171, 189-195 (1990)) as well as [3H]-dimethylstaurosporine (Gross et al, Biochem. Pharmacol., July 15;40(2):343-50 (1990)) bind reversibly to PKC-alpha.
The following U.S. Pat. No. 4,378,428, issued Mar. 29, 1983; U.S. Pat. No. 4,708,929, issued Nov. 24, 1987; U.S. Pat. No. 5,037,735, issued Aug. 6, 1991; U.S. Pat. No. 5,106,950, issued Apr. 21, 1992; U.S. Pat. No. 5,362,625, issued Nov. 8, 1994; U.S. Pat. No. 5,464,747, issued Nov. 7, 1995; U.S. Pat. No. 5,604,091, issued Feb. 18, 1997; U.S. Pat. No. 5,643,734, issued Jul. 1, 1997; U.S. Pat. No. 6,270,964, issued Aug. 7, 2001; and U.S. Pat. No. 6,294,330, issued Sep. 25, 2001 describe related subject matter. Applications WO 96/19732 and WO 98/06648 describe assays using complementation of enzyme fragments, and WO 00/072011 and WO 98/09169 describe assays for kinases and phosphatases. All patents, patent applications and references cited herein are hereby incorporated by reference in their entirety.
None of the above references provide methods and protocols for rapid screening of a large number of different agents to determine the affinity of the agents for a particular target.
It is, therefore, a subject of this invention to provide such method and protocols which permits rapid screening of a large number of different agents for their affinity to bind to a particular target.
The invention provides systems, including methods and reagents, for identifying enzyme inhibitors. The systems employ a conjugate of an enzyme donor and a known inhibitor of a target enzyme, an enzyme acceptor that binds to the enzyme donor to form an active indicator-enzyme complex, and a substrate for the indicator enzyme. The assay is performed by combining a candidate inhibitor compound, the conjugate of the enzyme donor and known inhibitor, the enzyme acceptor, and the substrate under binding conditions, where the candidate compound competes with the conjugate for the target enzyme. By measuring the rate of product formation or substrate depletion catalyzed by the indicator enzyme, the inhibitory activity of the candidate compound can be determined. The methodology may be particularly applicable for target enzymes that have substrates or products that are difficult to synthesize and/or detect, such as kinases and phosphatases.